Abstract
The protozoan parasite, Trypanosoma brucei, is spread by the tsetse fly and causes trypanosomiasis in humans and animals. Both the life cycle and cell cycle of the parasite are complex. Trypanosomes have eleven cdc2-related kinases (CRKs) and ten cyclins, an unusually large number for a single celled organism. To date, relatively little is known about the function of many of the CRKs and cyclins, and only CRK3 has previously been shown to be cyclin-dependent in vivo. Here we report the identification of a previously uncharacterised CRK:cyclin complex between CRK12 and the putative transcriptional cyclin, CYC9. CRK12:CYC9 interact to form an active protein kinase complex in procyclic and bloodstream T. brucei. Both CRK12 and CYC9 are essential for the proliferation of bloodstream trypanosomes in vitro, and we show that CRK12 is also essential for survival of T. brucei in a mouse model, providing genetic validation of CRK12:CYC9 as a novel drug target for trypanosomiasis. Further, functional characterisation of CRK12 and CYC9 using RNA interference reveals roles for these proteins in endocytosis and cytokinesis, respectively.
Highlights
In eukaryotes, cyclin-dependent kinases (CDKs) are of fundamental importance for cell cycle progression [1,2,3] and play essential roles in regulating gene expression [4,5,6,7], autophagy [8] and neuronal function [9] as well as key roles in responding to stresses [10,11]
CYC9 associates with CRK12 in vivo To enable functional characterisation of CYC9, CYC9 was tagged at its C-terminus with a Tandem Affinity Purification (TAP) [37] tag (CYC9:TAP) in procyclic T. brucei to facilitate the identification of in vivo kinase partner(s) of CYC9
Mass spectrometry of elution 3 identified two peptides, KMDAIDEVVRL and KKLHEMGIIHRD, that corresponded to T. brucei CRK12, strongly suggesting that CYC9 binds to CRK12 in vivo
Summary
Cyclin-dependent kinases (CDKs) are of fundamental importance for cell cycle progression [1,2,3] and play essential roles in regulating gene expression [4,5,6,7], autophagy [8] and neuronal function [9] as well as key roles in responding to stresses [10,11]. A CDK may bind to more than one cyclin during the cell cycle, and is targeted to different substrates at different phases of the cell cycle. Budding yeast express just one major cell cycle CDK, CDC28, which binds to different cyclins to promote successive cell cycle transitions [2]. Over 20 CDKs and numerous cyclins have been identified in mammalian cells, with many able to compensate in the absence of others [1]
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